Anesth Analg 2004;99:103-107
© 2004 International Anesthesia Research Society
doi: 10.1213/01.ANE.0000118110.90277.D2
ANESTHETIC PHARMACOLOGY
4-Chloro-m-Cresol Cannot Detect Malignant Hyperthermia Equivocal Cells in an Alternative Minimally Invasive Diagnostic Test of Malignant Hyperthermia Susceptibility
Lukas G. Weigl, PhD*,
Carmen Ludwig-Papst, PhD
, and
Hans G. Kress, MD PhD*
Departments of *Anesthesiology and Intensive Care Medicine (B) and
Surgery, Medical University Vienna, Vienna, Austria
Address correspondence and reprint requests to Lukas G. Weigl, PhD, Medical University Vienna, Department of Anesthesiology and Intensive Care Medicine (B), Währinger Gürtel 18-20, A-1090 Vienna, Austria. Address e-mail to lukas.weigl{at}univie.ac.at
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Abstract
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Malignant hyperthermia (MH) is an inherited skeletal muscle disorder triggered by commonly used anesthetics. Mutated ryanodine receptors have been identified as molecular targets. The sensitivity of myotubes from individuals classified by the in vitro contracture test as MH susceptible (MHS), normal (MHN), and equivocal (MHEH) was assessed for the Ca2+-releasing activity of 4-chloro-m-cresol (4-CmC) and caffeine. In this study, we sought to determine whether 4-CmC can differentiate the MH status of an individual on the basis of the release of intracellular Ca2+, particularly in regard to MHEH diagnosis. Intracellular Ca2+ concentration was determined photometrically with Fura2. Regions of the ryanodine receptor 1 harboring most of the described MH mutations were sequenced from MHS and MHEH cells. One MH mutation (Gly2434Arg) was found in one MHS individual. Results of the caffeine-induced Ca2+ release in MHS and MHN cells correlated well with the in vitro contracture test results. MHS cells showed a higher sensitivity against caffeine and, to a lesser extent, against 4-CmC. Cells of MHEH individuals showed low sensitivities against both caffeine and 4-CmC, comparable to those of the MHN group. Therefore, with myotubes, caffeine was able to discriminate between MHS and MHN cells, but both caffeine and 4-CmC failed to detect MHEH cells.
IMPLICATIONS: For the diagnostic test of susceptibility to malignant hyperthermia (MH), 4-chloro-m-cresol (4-CmC) has been proposed. In our study with differentiated human skeletal muscle cells, however, 4-CmC, like caffeine, could not distinguish between cells from individuals tested in the in vitro contracture test as normal and MH equivocal.
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Introduction
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The diagnosis of malignant hyperthermia (MH) is based on the in vitro contracture test (IVCT) of muscle bundles usually obtained by biopsy from the vastus lateralis muscle. According to the standardized protocols of the European Malignant Hyperthermia Group, a patient is diagnosed as susceptible to MH (MHS; reacts to halothane and caffeine), normal (MHN; no reaction to halothane and caffeine), or equivocal (MHE; i.e., response to only caffeine [MHEC] or only halothane [MHEH]). The percentage of the MHE group differs considerably among various laboratories and reaches values up to 40% (1). In our laboratory, 12% of the individuals tested between 1996 and 2002 were diagnosed as MHEH. The reason for an MHEH diagnosis is unknown, but because of the low specificity of the IVCT, this test result is often considered false positive (1). However, for safety reasons, such cases are clinically treated as MHS. The IVCT has been designed to reach maximum sensitivity to prevent false-negative test results, and, indeed, the sensitivity of the IVCT is 99.0%, whereas the specificity reaches only 93.6% (2). Linkage studies implicate a high level of locus heterogeneity in MH (3), indicating the existence of altered proteins other than the ryanodine receptor 1 (RyR1). Those altered proteins could lead to a halothane-induced Ca2+ release in MHS muscle. Because caffeine is a specific activator of calcium release through the RyR (4), the reason for MHEH diagnosis may result from failure to activate calcium release from other sites that may be affected by halothane in MHEH individuals.
To increase the specificity of the IVCT, new diagnostic substances are under investigation. 4-Chloro-m-cresol (4-CmC) has been proposed to be such a potential substitute or supplement for caffeine in the IVCT and in other so-called minimally invasive tests for MH (5,6). 4-CmC has a higher potency than caffeine to release Ca2+ through the RyR (7) and thus acts similarly to caffeine but probably exerts its effect via a different binding site (8).
In this study, we investigated whether it is possible to increase the specificity of the IVCT in the case of an MHEH diagnosis with the help of the Ca2+ imaging technique and 4-CmC. We tried to characterize cultured myotubes from individuals diagnosed MHEH as MHN or MHS with 4-CmC. To detect possible genetic alterations in the RyR1, we also sequenced the regions of the complementary DNA (cDNA) that harbor most of the described MH mutations.
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Methods
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The study was approved by the ethics committee of our hospital, and waste material of muscle biopsy samples (100–350 mg) was obtained from individuals undergoing the IVCT for the diagnosis of MHS. Cultured cells from 14 individuals were used: 2 MHS, 6 MHE, and 6 MHN. In the MHEH group, three individuals tested were relatives and were included in this study because the IVCT results are likely to result from a hereditary genomic variation rather than being false positive. Other individuals were chosen randomly.
Human skeletal muscle cell culture was performed as already described (9). In brief, muscle tissue was cut into small pieces and digested in collagenase (Type NB 8, 0.5 mg/mL Hanks’ balanced salt solution; Serva, Heidelberg, Germany). The cell suspension was centrifuged twice, washed, resuspended, and finally seeded in growth medium (Ham’s F12 supplemented with 15% fetal calf serum, 10 ng/mL epidermal growth factor, 200 ng/mL insulin, 400 ng/mL dexamethasone, 0.5 mg/mL fetuin, 0.5 mg/mL bovine serum albumin, 7 mM glucose, 4 mM L-glutamine, 200 U/mL penicillin, 200 µg/mL streptomycin, and 2.5 µg/mL amphotericin B) (10) onto 50-mL cell culture flasks for proliferation. Cells were kept at 37°C under 2.5% CO2 in an incubator, grown close to confluency, and reseeded on 25-mm glass coverslips coated with fibronectin. Adherent cells were exposed to differentiation medium (Dulbecco’s modified Eagle’s medium supplemented with 5% horse serum, and 4 mM L-glutamine, 100 ng/mL insulin, and 0.1 µg/mL gentamicin) to promote fusion of satellite cells to myotubes in an incubator with 5% CO2.
For determination of Ca2+ concentration, cells were incubated 35 to 45 min in loading buffer (Tyrode’s solution with 7 µM Fura2/AM; [Molecular Probes, Eugene, OR] and 0.025% Pluronic) at 37°C. Coverslips were washed and placed into a perfusion chamber of a fluorescence microscope at 400x magnification. Only cells reacting to the depolarization solution HK (high potassium Tyrode’s solution with 60 mM KCl and 80 mM NaCl; no Ca2+ added) with an increase in intracellular Ca2+ concentration ([Ca2+]i) were used for experiments, assuming a skeletal muscle-specific excitation-contraction coupling. Fluorescence intensity was monitored at an emission wavelength of 510 nm by altering excitation wavelengths between 340 and 380 nm by using a monochromator (VisiTech, Sunderland, UK).
For measurement of [Ca2+]i, images were recorded with a sample interval of 1 to 5 s and analyzed with the QC 900 software (VisiTech). Resting [Ca2+]i was defined by the average of the first 10 data points before application of any substances. The value of a [Ca2+]i transient was determined by the peak value reached within the time of substance application.
Calibration of fluorescence signals to calculate [Ca2+]i was performed according to Grynkiewicz et al. (11) and Thomas and Delaville (12). Dose-response curves were assessed from experiments as shown in Figure 1A. Increasing concentrations of substances were applied to a cell, and [Ca2+]i values were measured. After complete washout of the substance, the next larger concentration was applied. Least-square fitting of dose-response curves to the Hill equation was performed with the SigmaPlot program (SPSS Inc., Erkrath, Germany). From each individual of the MHEH and MHS groups, at least five cells were tested for each substance, but more were tested if the variability to the substance treatment was high. For statistical analysis, analysis of variance and Fisher’s least significant difference procedure were used to discriminate between means. Data are given as mean ± SE.
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Figure 1. Effect of caffeine on intracellular Ca2+ concentration ([Ca2+]i) in myotubes. Cells were derived from primary cultures of individuals diagnosed as nonsusceptible to malignant hyperthermia (MHN), susceptible to halothane but not caffeine (MH equivocal; MHEH), and susceptible to both caffeine and halothane (MHS). A, C, and E, Time course of [Ca2+]i increase when cells were rinsed with increasing concentrations of caffeine. HK denotes depolarization-induced Ca2+ release by a solution with increased K+ concentration in the absence of extracellular Ca2+. Such cells were considered to be differentiated muscle cells with intact skeletal muscle-specific excitation-contraction coupling. The experiment shown in (E) was from a cell of the MHS1 individual with a confirmed Gly2434Arg mutation in the ryanodine receptor 1 (RyR1). B, D, and F, Dose-response relationships for the effect of caffeine on [Ca2+]i in myotubes of the different diagnostic groups. The curves were calculated from experiments as shown on the left. Data for each MHS individual were analyzed individually. Please note the condensed scale in (F).
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For mutation analysis, RNA was isolated from myotubes with the RNeasy kit from Qiagen (Vienna, Austria). RNA was then reverse-transcribed by using the SuperScript First-Strand cDNA Synthesis System (Invitrogen, Lofer, Austria) according to the protocols of the manufacturers. The cDNAs were used as templates for polymerase chain reaction (PCR). Oligonucleotide primers according to McCarthy (13) were used to amplify the regions between nucleotides –12 and 2011, 6696 and 7414, and 14474 and 15173. The corresponding amino acid sequences are 1–670, 2232–2471, and 4824 to the C-terminal end of the protein (MH Regions 1–3). For purification, the PCR products were digested with exonuclease I (1 U/µL) and shrimp alkaline phosphatase (0.2 U/µL; both from USB Corp., Cleveland, OH). Purified PCR products were cycle-sequenced with the ABI Prism BigDye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems, Foster City, CA), and the sequencing products were analyzed with an ABI Prism 310 genetic analyzer. Both strands of all PCR products were sequenced.
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Results
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For this study, biopsy samples from 14 individuals were used. Six of them were diagnosed MHN, 6 MHEH, and 2 MHS (termed MHS1 and MHS2). Mutational analysis was performed for the MHEH and MHS individuals. None of the RyR1 mutations tested for and linked to MH was found in the MHEH group. The MHS1 individual showed a heterozygous Gly2434Arg mutation caused by the exchange of nucleotide C 7303 to A. For this individual, a further variation at position 594 (G 
A) was found that did not lead to the change of an amino acid. This single-nucleotide variation was also found in one of the MHEH individuals. In addition, this respective MHEH individual also carried the single nucleotide polymorphism at position 1668 (A G). For the MHS2 individual, we were not able to identify a known MH mutation. However, this MHS individual carried the single nucleotide polymorphism A1668G that was already present in one of the MHEH individuals. The people found to share these variations are not related. Although the MHEH individuals represent a heterogeneous group, we pooled their data for the following reasons: 1) they share a common diagnosis, 2) the data of the Ca2+ release experiments did not reveal significant differences among individuals, and 3) mutation analysis did not show genetic alterations of the RyR1.
The resting [Ca2+]i was not significantly different between the diagnostic groups at the P = 0.05 level (Table 1). Rinsing the cells with Ca2+-free HK solution, a depolarization solution with increased [K+], led to an increase in [Ca2+]i (see Fig. 1A for the time course), indicating a skeletal muscle-type excitation-contraction coupling. A significantly higher Ca2+ release was detected only in MHS1 cells that carried the Gly2434Arg mutation (Table 1). Myotubes from the other MH individual, as well as MHEH cells, did not differ in the reaction to depolarization compared with MHN cells.
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Figure 2. Effect of 4-cloro-m-cresol (4-CmC) on intracellular Ca2+ concentration ([Ca2+]i) in myotubes. The effect of 4-CmC was assessed in analogy to the experiments with caffeine shown in Figure 1. A, C, and E, Time course of [Ca2+]i and changes due to application of 4-CmC. The cells for the experiment shown in (E) were from individual 1 with a diagnosis of malignant hyperthermia (MHS) who carried the Gly2434Arg mutation in the ryanodine receptor 1 (RyR1) gene. Occasionally the induced Ca2+ release showed a biphasic course, preferentially at larger concentrations of 4-CmC [150 and 500 µM (A) and 500 µM (C)]. This biphasic release was not restricted to a specific diagnostic group. B, D, and F, Dose-response curves of 4-CmC for the effect on [Ca2+]i. Dose-response curves for MHS individuals are marked MHS1 and MHS2. MHN = not susceptible to malignant hyperthermia; MHEH = equivocal response only with halothane.
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Application of increasing caffeine concentrations (1–30 mM) to myotubes resulted in dose-dependent increases of [Ca2+]i. The effect of 1 mM caffeine was small, whereas 30 mM caffeine induced a change in [Ca2+]i by 180 nM (Fig. 1, A and B). The median effective concentration (EC50) value for caffeine on MHN cells was determined to be 6.6 mM (Table 1). Almost identical results were obtained for the MHEH group (Fig. 1, C and D), with an EC50 value of 6.3 mM. MHS cells of either individual showed EC50 values for Ca2+ release (1.4 and 2.4 mM caffeine) that were significantly different from the MHN values (Fig. 1, E and F). Thus, the sensitivity of MHS cells to caffeine was augmented 3- to 5-fold when compared with MHN or MHEH cells. In addition, the caffeine dose-response curve of MHS1 myotubes had a steep slope with a Hill coefficient more than 4, leading to a maximum activation of Ca2+ release already at a concentration of 3 mM caffeine. MHS2 cells reacted to 3 mM caffeine with 67% and MHN cells with only 15% of the maximum Ca2+ release.
MHS1 myotubes carrying the Gly2434Arg mutation showed a more than threefold higher caffeine-induced Ca2+ release than the cells from the MHN group (602 versus 180 nM). The second MHS individual without a confirmed mutation in the RyR1 responded to caffeine with a maximum increase in [Ca2+]i of 255 nM, which was not different from control cells and was only slightly more than MHEH cells (196 nM; Table 1).
Compared with caffeine, 4-CmC was more potent in releasing Ca2+ from intracellular stores. In control cells, the EC50 value for 4-CmC was determined to be 64.5 µM (Fig. 2A). In contrast to caffeine, 4-CmC occasionally induced a biphasic course of Ca2+ release, preferentially at concentrations larger than 75 µM (Fig. 2, A and C). This phenomenon was not restricted to a specific diagnostic group. In MHN cells, the calculated maximum increase in [Ca2+]i was 240 nM (Fig. 2B) and was therefore comparable to the Ca2+ release caused by caffeine. As expected and as already shown for other mutations (14), MHS cells showed an increased sensitivity to 4-CmC when compared with MHN cells. This difference was statistically significant only for MHS1 cells with an increase in sensitivity to 43.8 µM 4-CmC (Fig. 2F, Table 1). MHEH cells were not more sensitive to 4-CmC than MHN cells (Fig. 2, C and D). The EC50 value for Ca2+ release by 4-CmC was 73.3 µM, with a maximum increase in [Ca2+]i of 350 nM.
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Discussion
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In this study, we assessed the value of an additional in vitro test with differentiated myotubes to improve the specificity of the IVCT with particular respect to an MHEH diagnosis. The response of [Ca2+]i to caffeine showed significant differences for MHS and MHN cells only. The EC50 value for caffeine in MHS cells was significantly less than in MHN or MHEH cells. Thus, the difference in sensitivity against caffeine between the diagnostic groups correlated well with the IVCT.
In MHS myotubes, the caffeine sensitivity of Ca2+ release was approximately fourfold increased when compared with MHN cells. The data obtained with cultured myotubes therefore exactly reflect the results of the IVCT. At a concentration of 3 mM, caffeine induced nearly full activation of Ca2+ release in MHS cells, whereas MHN and MHEH cells were affected only by approximately 15%. This confirms the suitability of cultured muscle cells as a test system for functional in vitro studies of Ca2+-related muscle disorders. However, to obtain reliable results, a substantial number of experiments is obligatory, because the variability between single-cell measurements even of the same individual was large. In 1 case, 17 cells had to be tested to determine the EC50 value for 1 substance, whereas in other cases 5 cells were sufficient to obtain a satisfactory result.
4-CmC has been suggested to supplement caffeine in the IVCT because of higher sensitivity of RyR1 to 4-CmC (8). In fact, myotubes of all three diagnostic groups showed high sensitivity against 4-CmC. However, in cultured myotubes, the shift in sensitivity between MHS and MHN cells was less pronounced with 4-CmC (with a factor of approximately 1.4) than with caffeine (3.5-fold). Furthermore, in cells with the mutated RyR1, the slope of the 4-CmC dose-response curve did not increase to the same extent as with caffeine. On the basis of the sensitivities of myotubes against caffeine or 4-CmC obtained from our experiments, caffeine must be clearly considered the pharmacological substance best suited for the distinction of MHS and MHN cells, at least in an in vitro test with cultured myotubes. In none of the cases tested was the sensitivity of MHEH cells against 4-CmC increased when compared with MHN cells. Thus, it is not possible to improve the specificity of the IVCT by additional cellular testing with the help of either 4-CmC or caffeine.
For the time being, equivocal test results have to be accepted. The search for RyR1-specific agents to reduce the number of equivocal IVCT results does not seem very promising, because such agents will be able to detect defects only on the RyR1 but not on any other potential target that may evoke anesthetic-induced disturbances of Ca2+ homeostasis in skeletal muscle. Like caffeine, 4-CmC acts specifically on the RyR1 and thereby may be able to reveal disorders directly related to the RyR1 but not to other potential targets.
In conclusion, our investigations demonstrate that single-cell Ca2+ imaging is a useful experimental tool to discriminate between MHN and MHS cells and that 4-CmC is not superior to caffeine for this purpose. We thereby also suggest using caffeine instead of 4-CmC in so-called minimally invasive tests for MHS.
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Acknowledgments
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Supported by the University Hospital of Vienna and the Department of Anesthesiology and Intensive Care Medicine (B). CL-P is supported by the Bürgermeisterfonds of the City of Vienna No. 2093.
The authors thank Martin Hohenegger for critical reading and commenting on the manuscript and Katja Mackinger and Gertrude Weberhofer for technical assistance.
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References
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Accepted for publication January 7, 2004.
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Abstract
Introduction
